Transcript Industrial Biotechnology

Industrial Biotechnology
Lecturer Dr. Kamal E. M. Elkahlout
Assistant Prof. of Biotechnology
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CHAPTER 5
Metabolic Pathways for the Biosynthesis of
Industrial Microbiology Products
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THE NATURE OF METABOLIC PATHWAYS
• Metabolic pathway can be defined as series of chemical
reactions involved in converting a chemical (or a
metabolite) in the organism into a final product.
• The final product can be a metabolite product
(biochemical compound) or/and the cells of the
organism itself.
• Anabolism: collective reactions lead to the formation of
a more complex substance. (Anabolic pathway).
• Catabolism: collective reactions lead to the formation
of a less complex substance. (Catabolic pathway).
• The intermediates compounds involved in a metabolic
pathway & the final product is known as the endproduct (see Fig. 5.1).
• Catabolic reactions mostly studied with glucose.
• Four pathways of glucose breakdown to pyruvic
acid (or glycolysis) are currently recognized.
• Catabolic reactions often furnish energy in the form
of ATP and other high energy compounds, which
are used for biosynthetic reactions.
• A second function of catabolic reactions is to
provide the carbon skeleton for biosynthesis.
• Anabolic reactions lead to the formation of larger
molecules some of which are constituents of the
cell.
• Amphibolic intermediates: Kinds of metabolic
intermediates which are derived from catabolism
and which are also available for anabolism.
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INDUSTRIAL MICROBIOLOGICAL PRODUCTS AS
PRIMARY AND SECONDARY METABOLITES
Products of Primary Metabolism
Primary metabolism: Inter-related group of
reactions within a microorganism which are
associated with growth and the maintenance of life.
It is essentially the same in all living things and is
concerned with the release of energy, and the
synthesis of important macromolecules such as
proteins, nucleic acids and other cell constituents.
Stooping of primary metabolism causes death.
• Production of primary metabolites occurs in the
logarithmic phase of growth in a batch culture.
• Some of primary metabolites are cleared in (Table
5.1).
• Products of Secondary Metabolism
• Secondary metabolism, which was first observed in
higher plants, has the following characteristics:
• (i) It has no apparent function in the organism.
• The organism continues to exist if secondary
metabolism is blocked by a suitable biochemical
means.
• (ii) Secondary metabolites are produced in response
to a restriction in nutrients.
• They are produced after the growth phase, at the
end of the logarithmic phase of growth and in the
stationary phase (in a batch culture).
• They can be more precisely controlled in a
continuous culture.
• (iii) Secondary metabolism appears to be restricted
to some species of plants and microorganisms (and
in a few cases to animals).
• The products of secondary metabolism also appear
to be characteristic of the species.
• (iv) Secondary metabolites usually have ‘bizarre’
and unusual chemical structures and several closely
related metabolites may be produced by the same
organism in wild-type strains.
• This latter observation indicates the existence of a
variety of alternate and closely-related pathways.
• (v) The ability to produce a particular secondary
metabolite, especially in industrially important strains is
easily lost.
• This phenomenon is known as strain degeneration.
• (vi) Owing to the ease of the loss of the ability to
synthesize secondary metabolites, particularly when
treated with acridine dyes, exposure to high
temperature or other treatments known to induce
plasmid loss secondary metabolite production is
believed to be controlled by plasmids (at least in some
cases) rather than by the organism’s chromosomes.
• E. g., the case of leupeptin, in which the loss of the
metabolite following irradiation can be reversed by
conjugation with a producing parent.
• (vii) The factors which trigger secondary metabolism,
the inducers, also trigger morphological changes
(morphogenesis) in the organism.
• Inducers of Secondary Metabolites
• Autoinducers include the -butyrolactones (butanolides)
of the actinomycetes.
• The Nacylhomoserine lactones (HSLs) of Gramnegative
bacteria,
• The oligopeptides of Grampositive bacteria,
• BB-factor [3’-(1-butylphosphoryl)adenosine] of
rifamycin production in Amycolatopsis mediterrane.
• They function in development, sporulation, light
emission, virulence, production of antibiotics, pigments
and cyanide, plasmiddriven conjugation and
competence for genetic transformation.
• Of great importance in actinomycete fermentations
is the inducing effect of endogenous butyrolactones, e.g. Afactor (2-S-isocapryloyl-3Rhydroxymethyl--butyrolactone).
• A-factor induces both morphological and chemical
differentiation in Streptomyces griseus and
Streptomyces bikiniensis, bringing on formation of
aerial mycelia, conidia, streptomycin synthases and
streptomycin.
• Conidia can actually form on agar without A-factor
but aerial mycelia cannot.
• The spores form on branches morphologically
similar to aerial hyphae but they do not emerge
from the colony surface.
• In S. griseus, A-factor is produced just prior to
streptomycin production and disappears before
streptomycin is at its maximum level.
• It induces at least 10 proteins at the transcriptional
level.
• One of these is streptomycin 6phosphotransferase, an enzyme which functions
both in streptomycin biosynthesis and in resistance.
• In an A-factor deficient mutant, there is a failure of
transcription of the entire streptomycin gene
cluster.
• Many other actinomycetes produce A-factor, or related
α-butyrolactones, which differ in the length of the sidechain.
• In those strains which produce antibiotics other than
streptomycin, the α -butyrolactones induce formation
of the particular antibiotics that are produced, as well
as morphological differentiation.
• Microbial secondary metabolites include antibiotics,
pigments, toxins, effectors of ecological competition
and symbiosis, pheromones, enzyme inhibitors,
immunomodulating agents, receptor antagonists and
agonists, pesticides, antitumor agents and growth
promoters of animals and plants, including gibbrellic
acid, antitumor agents, alkaloids such as ergometrine, a
wide variety of other drugs, toxins and useful materials
such as the plant growth substance, gibberellic acid
(Table 5.2).
• They have a major effect on the health, nutrition,
and economics of our society.
• They often have unusual structures and their
formation is regulated by nutrients, growth rate,
feedback control, enzyme inactivation, and enzyme
induction.
• Regulation is influenced by unique low molecular
mass compounds, transfer RNA, sigma factors, and
gene products formed during post-exponential
development.
• The synthases of secondary metabolism are often
coded for by clustered genes on chromosomal DNA
and infrequently on plasmid DNA.
• Pathways of secondary metabolism are still not
understood to a great degree.
• Secondary metabolism is brought on by exhausion of a
nutrient, biosynthesis or addition of an inducer, and/or
by a growth rate decrease.
• These events generate signals which effect a cascade of
regulatory events resulting in chemical differentiation
(secondary
metabolism)
and
morphological
differentiation (morphogenesis).
• The signal is often a low molecular weight inducer
which acts by negative control, i.e. by binding to and
inactivating
a
regulatory
protein
(repressor
protein/receptor protein) which normally prevents
secondary metabolism and morphogenesis during rapid
growth and nutrient sufficiency.
TROPHOPHASE-IDIOPHASE RELATIONSHIPS IN
THE PRODUCTION OF SECONDARY PRODUCTS
• From studies on Penicillium urticae the terms
trophophase and idiophase were introduced to
distinguish the two phases in the growth of organisms
producing secondary metabolites.
• The trophophase (Greek, tropho = nutrient) is the
feeding phase during which primary metabolism
occurs.
• In a batch culture this would be in the logarithmic
phase of the growth curve.
• Following the trophophase is the idio-phase (Greek,
idio = peculiar) during which secondary metabolites
peculiar to, or characteristic of, a given organism are
synthesized.
• Secondary synthesis occurs in the late logarithmic,
and in the stationary, phase.
• It has been suggested that secondary metabolites
be described as ‘idiolites’ to distinguish them from
primary metabolites.
• ROLE OF SECONDARY METABOLITES IN THE
PHYSIOLOGY OF ORGANISMS PRODUCING THEM
• The theories in currency are discussed below; even
then none of these can be said to be water tight.
The rationale for examining them is that a better
understanding of the organism’s physiology will
help towards manipulating it more rationally for
maximum productivity.
• (i) The competition hypothesis: In this theory which
refers to antibiotics specifically,
• secondary metabolites (antibiotics) enable the
producing organism to withstand competition for food
from other soil organisms.
• In support of this hypothesis is the fact that antibiotic
production can be demonstrated in sterile and nonsterile soil, which may or may not have been
supplemented with organic materials.
• As further support for this theory is the wide
distribution of β-lactamases among microorganisms to
help these organisms to detoxify the β –lactam
antibiotics.
• The obvious limitation of this theory is that it is
restricted to antibiotics and that many antibiotics exist
outside Beta-lactams.
• (ii) The maintenance hypothesis: Secondary
metabolism usually occurs with the
• exhaustion of a vital nutrient such as glucose.
• It is therefore claimed that the selective advantage of
secondary metabolism is that it serves to maintain
mechanisms essential to cell multiplication in operative
order when that cell multiplication is no longer
possible.
• Thus by forming secondary enzymes, the enzymes of
primary metabolism which produce precursors for
secondary metabolism therefore, the enzymes of
primary metabolism would be destroyed.
• In this hypothesis therefore, the secondary metabolite
itself is not important; what is important is the pathway
of producing it.
• (iii) The unbalanced growth hypothesis: Similar to
the maintenance theory, this
• hypothesis states that control mechanisms in some
organisms are too weak to prevent the over
synthesis of some primary metabolites.
• These primary metabolites are converted into
secondary metabolites that are excreted from the
cell.
• If they are not so converted they would lead to the
death of the organism.
• (iv) The detoxification hypothesis: This hypothesis
states that molecules accumulated in the cell are
detoxified to yield antibiotics.
• This is consistent with the observation that the
penicillin precursor penicillanic acid is more toxic to
Penicillium chrysogenum than benzyl penicillin.
• Nevertheless not many toxic precursors of
antibiotics have been observed.
• (v) The regulatory hypothesis: Secondary
metabolite production is known to be associated
with morphological differentiation in producing
organisms.
• In the fungus Neurospora crassa, carotenoids are
produced during sporulation.
• In Cephalosporium acremonium, cephalosporin C is
produced during the idiophase when arthrospores
are produced.
• Numerous examples of the release of secondary
metabolites with some morphological
differentiation have been observed in fungi.
• Production of peptide antibiotics by Bacillus spp.
and spore formation.
• Both spore formation and antibiotic production are
suppressed by glucose; non-spore forming mutants
of bacilli also do not produce antibiotics.
• Reversion to spore formation is accompanied by
antibiotic formation has been observed in
actinomycetes.
• Production of gramicidin in sporulation of Bacillus spp.
• The absence of the antibiotic leads to partial
deficiencies in the formation of enzymes involved in
spore formation, resulting in abnormally heat-sensitive
spores.
• Peptide antibiotics therefore suppress the vegetative
genes allowing proper development of the spores.
• Production of secondary metabolites is necessary to
regulate some morphological changes in the organism.
• It could be that some external mechanism triggers off
secondary metabolite production as well as the
morphological change.
• (vi) The hypothesis of secondary metabolism as
the expression of evolutionary reactions: Zahner
has put forth a most exciting role for secondary
metabolism.
• Both primary and secondary metabolism are
controlled by genes carried by the organism.
• Any genes not required are lost.
• According to this hypothesis, secondary metabolism
is a clearing house or a mixed bag of biochemical
reactions, undergoing tests for possible
incorporation into the cell’s armory of primary
reactions.
• Any reaction in the mixed bag which favorably affects
any one of the primary processes, thereby fitting the
organism better to survive in its environment, becomes
incorporated as part of primary metabolism.
• According to this hypothesis, the antibiotic properties
of some secondary metabolites are incidental and not a
design to protect the microorganisms.
• This hypothesis implies that secondary metabolism
must occur in all microorganisms since evolution is a
continuing process.
• The current range of secondary metabolites is limited
only by techniques sensitive enough to detect them.
PATHWAYS FOR THE SYNTHESIS OF
PRIMARY AND SECONDARY
METABOLITES OF
INDUSTRIAL IMPORTANCE
• The main source of carbon and energy in industrial
media is carbohydrates.
• In recent times hydrocarbons have been used.
• The catabolism of these compounds will be
discussed briefly because they supply the carbon
skeletons for the synthesis of primary as well as for
secondary metabolites.
• The inter-relationship between the pathways of
primary and the secondary metabolism will also be
discussed briefly.
• Catabolism of Carbohydrates
• Four pathways for the catabolism of carbohydrates
up to pyruvic acid are known.
• All four pathways exist in bacteria, actinomycets
and fungi, including yeasts.
• The four pathways are the Embden-MeyerhofParnas, the Pentose Phosphate Pathways, the
Entner Duodoroff pathway and the
Phosphoketolase.
• These pathways are for the breakdown of glucose.
• Other carbohydrates easily fit into the cycles.
• (i) The Embden-Meyerhof-Parnas (EMP Pathways):
The net effect of this pathway is
• to reduce glucose (C6) to pyruvate (C3) (Fig. 5.2).
• Can be operate under both aerobic and anaerobic
conditions.
• Under aerobic conditions it usually functions with
the tricarboxylic acid cycle which can oxidize
pyruvate to CO2 and H2O.
• Under anaerobic conditions, pyruvate is fermented
to a wide range of fermentation products, many of
which are of industrial importance (Fig. 5.3).
• (ii) The pentose Phosphate Pathway (PP): This is
also known as the Hexose
• Monophosphate Pathway (HMP) or the
phosphogluconate pathway.
• EMP pathway provides pyruvate, a C3 compound,
as its end product, there is no end product in the
PP pathway.
• It provides a pool of triose (C3) pentose (C5),
hexose (C6) and heptose (C7) phosphates.
• The primary purpose of the PP pathway to generate
energy in the form of NADPA2 for biosynthetic and
other purposes and pentose phosphates for
nucleotide synthesis (Fig. 5.4)
• (iii) The Entner-Duodoroff Pathway (ED):
• The pathway is restricted to a few bacteria
especially Pseudomonas, but it is also carried out by
some fungi. It is used by some
• organisms in the enaerobic breakdown of glucose
and by others only in gluconate
• metabolism (Fig. 5.5)
• (iv) The Phosphoketolase Pathway: In some
bacteria glucose fermentation yields lactic acid,
ethanol and CO2.
• Pentoses are also fermented to lactic acid and
acetic acid.
• An example is Leuconostoc mesenteroides (Fig. 5.6).
• Pathways used by microorganisms
• The two major pathways used by microorganisms for
carbohydrate metabolism are the EMP and the PP pathways.
• Microorganisms differ in respect of their use of the two
pathways.
• Saccharomyces cerevisae under aerobic conditions uses
mainly the EMP pathway; under anaerobic conditions only
about 30% of glucose is catabolized by this pathway.
• In Penicillium chrysogenum, however, about 66% of the
glucose is utilized via the PP pathway.
• The PP pathway is also used by Acetobacter, the acetic acid
bacteria.
• Homofermentative bacteria utilize the EMP pathway for
glucose breakdown.
• The ED pathway is especially used by Pseudomonas.
• The Catabolism of Hydrocarbons
• Compared with carbohydrates, far fewer organism
appear to utilize hydrocarbons.
• Hydrocarbons have been used in single cell protein
production and in amino-acid production among
other products.
• (i) Alkanes: Alkanes are saturated hydrocarbons
that have the general formula
• C2 Hn+2. When the alkanes are utilized, the terminal
methyl group is usually oxidized to the
corresponding primary alcohol thus:
• The alcohol is then oxidized to a fatty acid, which then
forms as ester with coenzyme A.
• Thereafter, it is involved in a series of -oxidations (Fig.
5.7) which lead to the step-wise cleaving off of acetyl
coenzyme A which is then further metabolized in the
Tricarboxylic Acid Cycle.
• (ii) Alkenes: The alkenes are unsaturated
hydrocarbons and contain many double bonds.
• Alkenes may be oxidized at the terminal methyl group
as shown earlier for alkanes.
• They may also be oxidized at the double bond at the
opposite end of the molecule by molecular oxygen
given rise to a diol (an alcohol with two –OH
• groups).
• Thereafter, they are converted to fatty acid and utilized
as indicated above.
CARBON PATHWAYS FOR THE FORMATION
OF SOME INDUSTRIAL PRODUCTS
DERIVED FROM PRIMARY METABOLISM
The broad flow of carbon in the formation of
industrial products resulting from primary
metabolism may be examined under two
headings:
(i) catabolic products resulting from
fermentation of pyruvic acid and
(ii) anabolic products.
• Catabolic Products
• Derived from pyruvic acid produced via the EMP, PP, or
ED pathway.
• E.g., ethanol, acetic acid, 2, 3-butanediol, butanol,
acetone and lactic acid (Fig. 5.3).
• The nature of the products depends on the species of
organism & on the prevailing environmental conditions
(pH, temperature, aeration, etc).
• Anabolic Products
• Include amino acids, enzymes, citric acid, and nucleic
acids.
• The carbon pathways for the production of anabolic
primary metabolites will be discussed as each product
is examined.
CARBON PATHWAYS FOR THE
FORMATION OF SOME PRODUCTS OF
MICROBIAL SECONDARY METABOLISM
OF INDUSTRIAL IMPORTANCE
• The unifying features of the synthesis of secondary
metabolic products by microorganisms can be
summarized thus:
• (i) conversion of a normal substrate into important
intermediates of general metabolism;
• (ii) the assembly of these intermediates in unusaul
special mechanism;
• (iii) these special mechanisms while being peculiar
to secondary metabolism are not unrelated to
general or primary mechanism;
• (iv) the synthetic activity of secondary metabolism
appears in response to conditions favorable for cell
multiplication.
• Secondary metabolites are diverse in chemical
nature as well as in the organism which produce
them,
• They use only a few biosynthetic pathways which
are related to, and use the intermediates of, the
primary metabolic pathways.
• Based on the broad flow of carbon through primary
metabolites to secondary metabolites, (depicted in
Fig. 5.8) the secondary metabolites may then be
classified according to the following six metabolic
pathways.
• (i) Secondary products derived from the intact glucose
skeleton:
• The entire basic structure of the secondary product
may be derived from glucose as in streptomycin or
• form the glycoside molecule to be combined with a
non-sugar (aglycone portion) from another biosynthetic
route.
• The incorporation of the intact glucose molecule is
more common among the actinomycetes than among
the fungi.
• (ii) Secondary products related to nucleosides:
• The pentose phosphate pathway provides ribose for
nucleoside biosynthesis.
• Many secondary metabolites in this group are
antibiotics and are produced mainly by actinomycetes
and fungi. (nucleoside antibiotics such as bleomycin).
• (iii) Secondary products derived through the ShikimateChorismate Pathway:
• Shikimic acid (C7) is formed by the condensation of
erythrose-4- phosphate (C4) obtained from the PP pathway
with phosphoenolypyruvate (C3) from the EMP pathway.
• It is converted to chorismic acid which is a key intermediate in
the formation of numerous products including aromatic
aminoacids, such as phynylalamine, tryrosine and tryptophan.
• Chorismic acid is also a precursor for a number of secondary
metabolites including chloramphenicol, p-amino benzoic acid,
phenazines and phyocyanin which all have anticrobial
properties (Fig. 5.9).
• The shikimate-chorismate pathway is important for the
formation of aromatic secondary products in the bacteria and
actinomycetes.
• E. g., chloramphenicol and novobiocin.
• The route is less used in fungi, where the polyketide pathway
is more common for the synthesis of aromatic secondary
products.
• (iv) The polyketide pathway:
• Polyketide biosynthesis is highly characteristic of the
fungi, where more secondary metabolites are produced
by it than by any other.
• Most of the known polyketide-derived natural products
have been obtained from the fungi.
• The addition of CO2 to an acetate group gives a
malonate group.
• The synthesis of polyketides is very similar to that of
fatty acids.
• In the synthesis of both groups of compounds acetate
reacts with malonate with the loss of CO2.
• By successive further linear reactions between the
resulting compound and malonate, the chain of the
final compound (fatty acid or polyketide) can be
successively lengthened.
• Due to this a chain of ketones or a -polyketomethylene
(hence the name polyketide) is formed (Fig. 5.10).
• The polyketide ( β- poly-ketomethylene) chain made up
of repeating C-CH2 or ‘C2 units’, is a reactive proteinbound intermediate which can undergo a number of
reactions, notably formation into rings.
• Polyketides are classified as triketides, tetraketides,
pentaketides, etc., depending on the number of ‘C2
units’.
• Thus, orsellenic acid which is derived from the straight
chain compound in Fig. 5.11 with four ‘C2-units’ is a
tetraketide.
• Although the polyketide route is not common in
actinomycetes, a modified polyketide route is used in
the synthesis of tetracyclines by Streptomyces griseus.
• (v) Terpenes and steroids:
• The second important pathway from acetate is that
leading via mevalonic acid to the terpenes and steroids.
• Microorganisms especially fungi and bacteria
synthesize a large number of terpenes, steroids,
carotenoids and other products following the ‘isoprene
rule’.
• These compounds are all derivatives of isoprene, the
five-carbon compound.
• Simply put the isoprene rules consist of the following
(Fig. 5.12):
• (i) Synthesis of mevalonate from acetate or leucine
• (ii) Dehydration and decarboxylation to give isoprene
followed by condensation to give isoprenes of various
lengths.
• (iii) Cyclization (ring formation) e.g., to give steroids.
• (iv) Further modification of the cyclised structure.
• The route leads to the formation of essential steroid
hormones of mammals and to a variety of secondary
metabolites in fungi and plants.
• It is not used to any extent in the actinomycetes.
• vi) Compounds derived from amino acids:
• Intermediates from glucose catabolism can introduce
prcursores for amino acid synthesis.
• Serine (C3N) and glycine (C2N) are derived from the
triose (C3) formed glucose; valine (C5N) is derived from
acetate (C3); aspartatic acid (C4N) is derived from
oxeloacetic acid (C4) while glutamic acid (C5N) is derived
from oxoglutamic acid (C5) (Fig. 5.13) .
• Aromatic amino acids are derived via the shikimic
pathway.
• Secondary products may be formed from one, two or
more amino acids.
• E.g., (with one amino acid group) is hadacidin which
inhibits plant tumors and is produced from glycine and
produced by Penicillium frequentants according to the
formula shown below:
• E.g., (with two or more amino acid group) Other
examples are the insecticidal compound, ibotenic acid
(Amanita factor C) produced by the mushroom Amanita
muscaria and psilocybin, a drug which causes
hallucinations and produced by the fungus Psiolocybe
(Fig. 5.14), the ergot alkaloids produced by Clavicepts
purpureae also belong in this group as does the
antibiotic cycloserine.
• Among the secondary products derived from two
amino acids are gliotoxin which is produced by
members of the Fungi Imperfecti, especially
Trichoderma and which is a highly active anti-fungal
and antibacterial (Fig. 5.14) and Arantoin, an
antiviral drug also belongs to this group.
• The secondary products derived from more than
two amino acids include many which are of
immense importance to man.
• These include many toxins from mushrooms e.g the
Aminita toxins (Fig. 5.15) (phalloidin, amanitin)
peptide antibiotics from Bacillus spp and a host of
other compounds.
• An example of a secondary metabolite produced
from three amino acids is malformin A (Fig. 5.15)
which is formed by Aspergillus spp.
• It induces curvatures of beam shoots and maize
seedlings. It is formed from L-leucine, D-leucine,
and cysteine.